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Numerous chemotrophic microorganisms inhabit high-temperature (> 65 °C) systems of Yellowstone National Park (WY, USA). Prior geochemical and metagenome characterization has identified the primary electron donors and acceptors and phylotypes distributed across a range in pH and geochemical conditions. Although several chemolithoautotrophs are expected to play a direct role in the fixation of inorganic C in these communities, little work has directly identified the importance of this process in situ. Consequently, the primary goal of this thesis was to evaluate the role of CO 2 fixation across numerous types of geothermal habitats and to explore autotroph-heterotroph interactions that may control community composition. Genes encoding enzymes for inorganic C fixation pathways were identified in assembled genome sequence corresponding to the predominant autotrophs (Crenarchaeota and Aquificales) observed in Fe(III)-oxide mats, sulfur sediments, and filamentous streamer communities. Carbon isotope (13 C) mixing models were used to interpret the 13C compositional values of microbial samples as a function of 13C-dissolved inorganic C (DIC) and 13 C-organic C (DOC and/or landscape sources). The relative abundance of autotrophs versus heterotrophs identified in complementary metagenome analysis and respective CO 2-fixation fractionation factors were utilized in site-specific mixing models to calculate minimum contributions of DIC-derived microbial C across 15 different microbial communities. Genome sequence was also used to develop stoichiometric reaction networks for a primary autotroph (Metallosphaera yellowstonensis) and heterotroph ('Geoarchaeota') important in acidic Fe(III)-oxide mats. Possible modes of biomass production were evaluated for different C sources and/or electron donors as a function of oxygen cost. The total oxygen flux was also used to predict the rate of Fe(II)-oxidation, and these values were compared to Fe(III)-oxide deposition rates and oxygen fluxes measured in situ. Stoichiometric modeling and elementary flux mode analysis established an optimum autotroph to heterotroph ratio (2.4:1) for DIC-derived biomass dependent on Fe(II) as the electron donor. Comparison of predicted Fe(II)-oxidation rates with observed Fe(III)-oxide deposition rates and oxygen flux measurements using microelectrodes suggest the importance of other oxygen consuming processes. Results from this thesis demonstrated the importance of inorganic C fixation in numerous geochemically distinct high-temperature microbial habitats, and the potential for DIC-derived biomass to support other hyperthermophilic heterotrophic organisms.